Uric acid test procedure

ABSTRACT

TEST PROCEDURE WHICH IS USEFUL IN THE DETERMINATION OF URIC ACID IN BODY FLUIDS. THE TEST REAGENT COMPRISES A SOLUTION OF 2,4-DIMETHYLQUINOLINE IN ETHYL ALCOHOL IN THE PRESENCE OF SULFURIC ACID. TEST REAGENT TURNS BROWN IN THE PRESENCE OF URIC ACID AND CAN BE USED FOR THE QUALITATIVE AND QUANTITATIVE DETERMINATION OF URIC ACID.

United States Patent M 3,554,701 URIC ACID TEST PROCEDURE William N.Cottrell, Jr., Indianapolis, Ind., assignor to Bio-Dynamics, Inc.,Indianapolis, Ind., a corporation of Indiana No Drawing. Filed Jan. 23,1969, Ser. No. 793,580 Int. Cl. C07d 33/00; G01n 33/ 1 6 US. Cl. 23-43010 Claims ABSTRACT OF THE DISCLOSURE Test procedure which is useful inthe determination of uric acid in body fluids. The test reagentcomprises a solution of 2,4-dimethylquinoline in ethyl alcohol in thepresence of sulfuric acid. Test reagent turns brown in the presence ofuric acid and can be used for the qualitative and quantitativedetermination of uric acid.

BACKGROUND OF THE INVENTION Uric acid is normally present in the bodyfluids of warm blooded animals. For diagnostic purposes it is oftendesirable to ascertain the uric acid level of certain body fluids. Inaccordance with this invention the uric acid content of body fluids canbe readily and inexpensively determined.

The metabolism of warm blooded animals produces uric acid in varyingamounts from ingested foods. In a given warm blooded animal the uricacid content of a body fluid must be held within carefully prescribedlimits can cause severe problems, it is desirable to have a quick andeasy test for the determination of uric acid in body fluids. The priorart includes quantitative and qualitative tests for the determination ofuric acid in body fluids. However, most prior art test procedures dependon the fact that the enzyme uricase quantitatively and qualitativelydegrades uric acid. Hence, these prior art testing procedures areenzymatic in nature. Because these prior art testing procedures areenzymatic the variables in the test must be carefully controlled andthey require a definite incubation period in which to allow the enzymeuricase to function.

In contrast with this prior art, the test procedure of this invention isnot enzymatic and gives a direct reading. The general test procedure ofthis invention is colori- Patented Jan. 12, 1971 metric 'in nature andhence can be used as a qualitative and quantitative indicator for thepresence of uric acid. While many of the prior art test procedures, dueto the fact that they are enzymatic, require a definite incubationperiod, the test procedure of this invention does not require anincubation period and gives a reading immediately. Likewise, due to thefact that the test procedure of this invention is quick and relativelysimple, it can be performed directly in the doctors office without theneed for taking the patents sample, sending the sample out for analysisand waiting for an extended period of time for the results of theanalysis. Accordingly, in an emergency situation, the fact that the testprocedure of this invention can be performed quickly can be verysignificant.

SUMMARY OF THE INVENTION This invention is concerned With a testprocedure for uric acid in body fluids. The test procedure of thisinvention comprises adding to a sample of a body fluid a solution of2,4-dimethy1quinoline in ethyl alcohol. After this addition the pH ofthe solution is lowered by the addition of sulfuric acid. Upon theaddition of sulfuric acid a brown color develops in the presence of uricacid. This brown color absorbs strongly in the region of 532,, andaccordingly can be used as a qualitative and quantitative indicator forthe presence of uric acid.

The primary object of this invention is a superior test procedure forthe presence of uric acid in body fluids.

Another object of this inventon is a non-enzymatic test procedure foruric acid in body fluids. 1

Still another object of this invention is a test procedure for uric acidwhich will give an instantaneous reading.

Finally the objects of this invention include all the other novelfeatures which will be obvious from the specification and claims athand.

DESCRIPTION OF THE PREFERRED EMBODIMENT The subject invention relates toa process wherein a solution of 2,4-dimethylquinoline in ethyl alcoholis used as a colorimetric indicator for uric acid in the presence ofsulfuric acid. When the solution of 2,4-dimethylquinoline is added to abody fluid containing uric acid and sulfuric acid is added thereto theresulting solution turns brown.

For use in accordance with this invention, the concentration of2,4-dimethylquinoline in ethyl alcohol can be from about 1% to about 10%by volume. A more preferred range for this concentration is from about1% to about 5%, while a most preferred concentration is 2%.

When the solution of 2,4-dimethylquinoline in ethyl alcohol is added toa body fluid and the pH of the solution is lowered with sulfuric acid, abrown complex is formed in accordance with Equation 1 below. It is to benoted that while applicant is not sure of the exact mechanism wherebythe brown complex is produced, it is thought that Equation 1 accuratelyrepresents the process of this invention.

Equation 1 o H H J CINE.

l H OH: H

For purposes of developing the brown complex a stoichiometric amount of2,4-dimethylquinoline in ethyl alcohol is needed as based on the amountof uric acid present in the body fluid sample. While a stoichiometricamount of the test reagent is all that is needed in accordance with thisinvention, it is desirable to utilize an excess of the test reagent.Generally, it is preferred that an excess on the order of to percentover the stoichiometric amount be used for purposes of developing thebrown color in accordance with this invention.

After the solution of 2,4-dimethylquinoline in ethyl alcohol is added tothe body fluid sample, the pH of the composite solution is lowered tothe range of from about .1 to 3 by the addition of concentrated sulfuricacid. For use in accordance with this invention, concentrated sulfuricacid is added until the concentration of sulfuric acid in the overallsolution is on the order of 30 to 50 percent. A more preferred range forthis concentration is from about to about percent, while a mostpreferred concentration for use in accordance with this invention is 49percent.

It is to be noted that for use in accordance with this invention the pHof the composite solution must be lowered with sulfuric acid. That is,the color reaction of this invention is not strictly a function of pH,but instead it is thought that the sulfate ions produced by the sulfuricacid have a part in the reaction and aid in the formation of the browncomplex of this invention. Due to this fact, sulfuric acid must beutilized as a pH lowering medium.

The test procedure of ths invention can be carried out at any convenienttemperature. However, it is preferred that the test of this invention becarried out at room temperature.

The subject test can be carried out on many types of body fluids. Forexample, as a starting material the test of this invention can utilizeblood serum, plasma, urine, etc. Likewise, the invention is applicableto standard uric acid solutions such as solutions of uric acid in abuffered acidic medium.

It is within the purview of this invention to add to the compositions ofthis invention compatible materials which do not affect the basic andnovel characteristics of the composition of this invention. Among suchmaterials are coloring agents, including dyes and pigments, and similaradditives. Additives such as antioxidants, stabilizers and antifoaming,may also be aded. The upper limit of the quantity of additives isusually about 2 weight percent of the product.

N CH3 l 1; t

The following examples will illustrate the subject invention. Theseexamples are given for the purpose of illustration and not for purposesof limiting this invention. (All parts percent are given by weightunless otherwise specified.)

EXAMPLE I A reagent was prepared by adding 1 ml. of2,4-dimethylquinoline to 100 ml. of ethanol (Formula No. 3). Theresulting solution was continuously stirred until the2,4-dimethylquinoline was completely dissolved. Whereupon the solutionwas covered to prevent evaporation and protected from light.

To a small vial was added .25 ml. of the above described reagent.Likewise .01 ml. of blood serum was added. To the resulting solution wasadded 0.25 ml. of 98% concentrated sulfuric acid. The resulting solutionwas mixed and allowed to cool slightly.

Upon the addition of the concentrated sulfuric acid the solution turneddark brown indicating the presence of uric acid in the blood serum. Areading was taken at 532p. on a Coleman Colorimeter Model 124 asproduced by the Coleman Instrument Co. This reading indicated that theconcentration of uric acid in the blood serum was 4.5 gram percent. Thisis to be compared with the concentration of the serum sample which was4.6 gram percent.

EXAMPLE II Using the reagent and test procedure of Example I, the testwas repeated using hydrochloric acid in lieu of sulfuric acid. Upon theaddition of the hydrochloric acid an immediate precipitate was formedand there was no colorimetric reading in the range of 532g. Due to thefact that an immediate precipitate was formed, concentrated hydrochloricacid is not an acceptable substitute for the sulfuric acid of thisinvention.

EXAMPLE III Using the reagent and test procedure of Example Iconcentrated nitric acid was substituted for the concentrated sulfuricacid. Upon the addition of the concentrated nitric acid, nitrous oxidewas formed and was emitted from the solution in the form of a brown gas.The resulting solution did not absorb in the region of 532 Due to thefact that the solution did not absorb in this range and the fact thatnitrous oxide was formed,

this test procedure could not be utilized as an analytical tool for thedetermination of uric acid.

EXAMPL'E IV Using the reagent and test procedure of Example Iconcentrated acetic acid was substituted for the concentrated sulfuricacid of Example I. No reaction occurred and the solution did not absorbin the range of 53211.. Because it did not absorb in this range,concentrated acetic acid cannot be substituted for the concentratedsulfuric acid of this invention.

EXAMPLE V Using the reagent and test procedure of Example I the uricacid content of a human serum sample was determined by' the absorptionin the range of 532,11 which indicated that the sample had a uric acidcontent of 4.8 gram percent. This is to be compared with the knownconcentration of the serum sample in question which was 4.9 grampercent.

EXAMPLE VI Using the reagent and test procedure of Example I uric acidcontent of a serum control was determined. The absorption in the rangeof 532,11. was indicative of the fact that the uric acid content of theserum control was 10.3 gram percent. This is to be compared with theknown uric acid content of the sample which was 10.5 gram percent.

EXAMPLE VII A standard uric acid solution was prepared by dissolvinggrams of uric acid (C.P. grade) in 70 ml. of distilled water whereupon 5ml. of concentrated hydrochloric acid was added and the solution wasdiluted to 100 ml. with distilled water. Using the reagent and testprocedure of Example I, it was determined that the percent transmissionsof various concentrations of the standard solution are in accordancewith Table I. From Table I, it can be seen that the percent transmissionis a function of the uric acid concentration. With the data of Table I astandard curve can be set up which can be used in unknowndeterminations.

TABLE I Uric acid concentration in gram percent:

Percent transmission 43 O-PUIG What is claimed is:

1. A test reagent for uric acid in body fluids com prising sulfuric acidand a solution of 2,4-dimethylquinoline in ethyl alcohol.

2. The test reagent of claim 1 wherein the concentration of2,4-dimethylquinoline in ethyl alcohol is from about 1 to about 10percent by volume.

3. The test reagent of claim 1 wherein the concentration of2,4-dimethylquinoline in ethyl alcohol is from about 1 to about 5percent by volume.

4. The test reagent of claim 1 wherein the concentration of2,4-dimethylquinoline in ethyl alcohol is 2 percent by volume.

5. A testing procedure for the determination of uric acid in a bodyfluid which comprises the steps of bringing said body fluid into contactwith a solution of 2,4- dimethylquinoline in ethyl alcohol and addingthereto sulfuric acid.

6. The process of claim 5 wherein the concentration of2,4-dimethylquinoline in ethyl alcohol is from about 1 to about 10percent by volume, and the concentration of sulfuric acid in the overalltest solution is from about 30 to about percent.

7. The process of claim 5 wherein the concentration of2,4-dimethylquinoline in ethyl alcohol is from about 1 to about 5percent by volume, and the concentration of sulfuric acid in the overalltest solution is from about 45 to about 50 percent.

8. The process of claim 6 wherein the concentration of2,4-dimethylquinoline in ethyl alcohol is 2 percent by volume andwherein the concentration of sulfuric acid in the overall test solutionis 49 percent.

9. The process of claim 6 wherein a stoichiometric excess of from about30 to about 40 percent of 2,4-dimethylquinoline in ethyl alcoholsolution is used as based on the stoichiometric relationship of thereaction of 2,4- dimethylquinoline with uric acid.

10. The process of claim 6 wherein a .01 ml. portion of a body fluid isutilized in conjunction with .25 ml. of a solution of a 1 percent byvolume of 2,4-dimethylquinoline in ethyl alcohol and .25 ml. of 98percent concentration sulfuric acid which is added thereto, wherein thequalitative and quantitative uric acid content of said body fluid isdetermined by colorimetric deterrni nation at about 532 1..

References Cited UNITED STATES PATENTS 8/1967 Wachter 23-230BioX 2/1970Hughes 23230Bi0 MORRIS O. WOLK, Primary Examiner R. M. REESE, AssistantExaminer US. Cl. X.R. 252408; 260-283

